Eight of the 17 FDA-approved antiretroviral drugs for treating HIV-1 infection are NRTI such as AZT. NRTI are 3'-hydroxyl lacking analogs of the dNTP substrates used by HIV-1 RT, thus when an NRTI is added to the 3'-end of the nascent DNA chain, viral DNA synthesis is terminated. HIV-1 resistance to AZT (and other NRTI including abacavir) is a serious therapeutic problem and arises from RT-catalyzed phosphorolytic excision of the incorporated NRTI. Excision is enhanced by specific mutations in RT termed TAMs (thymidine analog mutations). After excision of the incorporated NRTI, viral DNA synthesis can resume to complete viral replication. Specific inhibitors of NRTI excision would be therapeutically useful by preventing removal of incorporated NRTI, thereby restoring activity of NRTI such as AZT and abacavir against TAM containing HIV-1. Optimally, such inhibitors should not reduce incorporation of NRTI as this would obviously antagonize their antiretroviral activity. We hypothesize that selective inhibitors of NRTI excision can be identified because NRTI incorporation and excision reactions use different substrates and have different rate limiting steps. Our preliminary data show certain bisphosphonate compounds may have utility in this context. We therefore propose (1) To screen and characterize an existing library of bisphosphonates. This includes development of a rapid primer unblocking screening assay, and detailed biochemical and virological analyses of screening hits; (2) To optimize the potency of bisphosphonate inhibitors of NRTI excision. This includes molecular modeling to predict the optimal bisphosphonate analog pharmacophore, and crystallographic studies of selected bisphosphonate analogs bound to covalent binary complexes of RT - template/AZT-primer; (3) To synthesize and characterize new inhibitor analogs predicted from Aim 2.; (4) To evaluate approaches to improve cell uptake of bisphosphonates. These include the use of drug carriers and chemical modification of bisphosphonates to improve cell uptake and antiviral activity.